Development Of Immunoassays For Escherichia Coli And Listeria Monocytogenes

Enterohemorrhagic Escherichia coli(EHEC)0157:H7 is a foodborne pathogen that causes severe outbreaks of infectious diseases,accounting for approximately 2-7%of 73,000 foodborne diseases worldwide.Listeria monicytogenes is a foodborne pathogen that can infect humans and animals.It can cause various diseases such as sepsis and meningitis while the mortality rate is as high as 30%.Traditional detection methods are time-consuming and laborious,and molecular biological detection methods are complicated to operate and prone to false positive results.Therefore,there is an urgent requirement to establish a rapid and stable detection method.In this study,a double-antibody sandwich ELISA assay was established for E.coli 0157:H7.The optimal detection conditions were:the chicken polyclonal antibody(IgY)was used as the capture antibody and the coating amount was 0.5 μg/well;the 1%skim milk was selected as the blocking solution and was blocked for 1.0 h;the rabbit polyclonal antibody(IgG)was used as the detection antibody,the dilution rate was 1:8000 and the incubation tiem was 1.0 h;the HRP-goat anti-rabbit antibody was applied for 0.5 h.The detection limit of the sandwich ELISA was 1.7×105 CFU/mL for pure bacterial culture and 101 CFU/mL and 100 CFU/mL for artificially enriched beef and milk samples after enrichment for 3 h at 37℃,respectively.To test the specificity of the sandwich ELISA,the other three E.coli 0157 strains,nine non-0157 E.coli strains and six other stains were used as controls.The results indicated that the EHEC 0157 strains showed similar reactions and other strains showed negative reactions.The colloidal gold test strips assay was established for E.coli 0157:H7.Gold nanoparticles(AuNPs)with 20-nm diameter crosslinked with 15 μL/mL IgG were coated on a conjugation pad The test line(T-line)was coated with 1.5 mg/mL IgG and the contest line(C-line)was coated with 5-fold diluted goat anti-rabbit antibody.The detection limit was determined as 105 CFU/mL for pure bacterial culture and 2.5 CFU/25g for artificially enriched beef and milk samples after enrichment for 6 and 7 h,respectively.In addition,an immunoassay assay was initially established for L.monocytogenes.Immunized with a L.monocytogenes surface protein,a hybridoma cell with good specificity was prepared by intraperitoneal injection and cell fusion.And a monoclonal antibody with a titer of 810000 and good specificity was obtained.The mouse monoclonal antibody was used as the capture antibody with coating amount 0.5 μg/well.The rabbit polyclonal antibody prepared previously was used as the detection antibody with dilution rate of 1:100.Skim milk powder with 0.5%concentration was selected as blocking buffer and blocked at 37℃ for 1.0 h;.The goat anti-rabbit antibody was incubation for 0.5 h.The detection limit of the detection was finally determined as 104 CFU/mL.Five different strains of L.monocytogenes,five different species of Listeria and five stains of non-Listeria were used to evaluate the specificity of the method and the result showed that the detection method had good specificity.